This invention relates to multipotent paramunity inducers based on combinations of poxvirus components, a method for preparing same, and their use as drugs.
The immune system of warm-blooded animals, especially of mammals and birds, consists of an antigen-specific part and an antigen non-specific part. The two parts are cross-linked and so form a uniform organic system. The antigen-specific mechanisms are responsible for building up an immunity, while the antigen non-specific are responsible for building up paramunity. Accordingly, for both historical and functional reasons the antigen non-specific part of the defence is known as the paraspecific immune system. Up until the present, immunology research has been mainly concerned with the antigen-specific part of the immune system, i.e. with how immunity is formed. The utilization of this defence potential led for example to the development of active and passive immunization. In contrast, the exploitation of the paraspecific activities of the immune system for prevention and cure is still in its early stages. The paraspecific immune system makes it possible for the organism to mount an immediate defence when confronted by the most diverse foreign substances, infectious pathogens, toxins and transformed cells of the organism itself. There are patterns of close functional interplay between the paraspecific and the specific activities of the immune system, generally involving a flow of information from the paraspecific mechanisms, which react first, to the specific mechanisms of the immune system. In this way, in cases where there is infection with particularly virulent pathogens the paraspecific defence of the organism is able to bridge the gap until specific immunity has had time to develop.
Since Edward Jenner introduced protective immunization against smallpox in 1798 using a vaccine obtained from animals (cattle or horses) and based on the vaccinia virus, empirically obtained results have been reported showing that protective immunization against smallpox resulted in those vaccinated recovering surprisingly rapidly, or without complications, from other infections and diseases, especially chronic and relapsing complaints, from which they happened to be suffering at the time of vaccination. In particular this applies to herpes infections of varying genesis, papillomas, chronic eczemas and pathological conditions of the ears, nose and throat. As well as this, however, it was noted that those immunized showed a generally raised short-term level of resistance to acute ambient infections. Similar phenomena have been noted after protective immunization against forms of animal pox. It was deduced from these findings that poxviruses or certain structural components of these viruses can positively influence the organism""s ability to resist infections and tumours, on a non-specific level. Because these non-specific healing processes commence immediately after vaccination and hence 5 to 7 days before the specific immunity conferred by vaccination develops, as well as parallel thereto, in 1978 A. Mayr designated these non-specific consequences of a prophylactic immunization as xe2x80x9cparaspecificxe2x80x9d. Accordingly, medicaments produced specifically to exploit such paraspecific effects are known as xe2x80x9cparamunity inducersxe2x80x9d; see A. Mayr, xe2x80x98Prxc3xa4munitxc3xa4t, Prxc3xa4munisierung und paraspezifische Wirkung von Schutzimpfungenxe2x80x99, Mxc3xcnch. Med. Wschr. 1978, Vol. 120, pp. 239 to 242; A. Mayr, H. Raettig, H. Stickl and M. Alexander, 1979: xe2x80x98Paramunitxc3xa4t, Paramunisierung, Paramunitxc3xa4tsinducerxe2x80x99, Fortschr. Med. 97, pp. 1159 to 1165 and 1205 to 1210.
DE-A-27 14 665 and U.S. Pat. No. 4,191,745 disclose preparations based on poxviruses for treating herpes zoster and other herpes infections. In veterinary medicine, paramunity inducers have been produced from purified, attenuated and inactivated avipox and parapox viruses. These paramunity inducers are registered in the European countries as xe2x80x9cDuphapindRxe2x80x9d and xe2x80x9cDuphamunRxe2x80x9d (PIND-AVI) and as xe2x80x9cBaypamunRxe2x80x9d (PIND-ORF) for virtually all species of farm and domestic animals. Paramunity inducer PIND-AVI is prepared from an attenuated avipox virus, strain HP-1, and paramunity inducer PIND-ORF is prepared from an attenuated parapox virus, strain D-1701. The attenuated viruses are rendered inactive in a manner known per se, e.g. by xcex3-radiation or chemical means such as treatment with xcex2-propiolactone.
The drugs with paramunizing properties which are preferred for use as immunostimulants and to provide xe2x80x9cadjuvant immunotherapyxe2x80x9d in human medicine are BCG (Bacillus Calmette Guerin), levamisole and Corynebacterium parvum (syn. Propionibacterium acnes). However, these preparations are inferior to the poxvirus-based paramunity inducers in terms of their paramunizing action, as may be seen from the results summarized in Table 1, which were obtained in tests in which these preparations were compared with PIND-AVI and PIND-ORF in the VSV baby mouse test; see A. Mayr, A. M. Bxc3xcttner, S. Pawlas, V. Erfle, B. Mayr, R. Brunner and K. Osterkorn, 1986: xe2x80x98Vergleichende Untersuchungen xc3xcber die immunstimulierende (paramunisierende) Wirksamkeit von BCG, Levamisol, Corynebacterium parvum und Prxc3xa4paraten aus Pockenviren in verschiedenen xe2x80x9cin vivoxe2x80x9dxe2x80x94und xe2x80x9cin vitroxe2x80x9dxe2x80x94Testenxe2x80x99, J. Vet. Med. B 33, pp. 321 to 339.
It is the object of the present invention to provide, for therapeutic applications in human and veterinary medicine, multipotent paramunity inducers based on poxvirus components, which have been improved in respect of their potency and in respect of their paramunization-related activities. It is a further object of the present invention to provide an improved method for preparing said multipotent paramunity inducers using a new technique of virus propagation in cell cultures and/or virus inactivation. Lastly, it is yet another object of the invention to provide pharmaceutical compositions for use as drugs based on said multipotent paramunity inducers.
The invention therefore relates to multipotent paramunity inducers based on a combination of two or more poxvirus components derived from various poxvirus strains having paramunizing properties. The invention further relates to multipotent paramunity inducers based on a combination of a plurality of different species of inactivated, attenuated poxviruses with paramunizing properties. The invention also relates to a method for preparing paramunity inducers in which two or more poxvirus components derived from different poxvirus strains with paramunizing properties are combined. Lastly, the invention relates to methods for preparing the multipotent paramunity inducers according to the invention, and to pharmaceutical compositions comprising said multipotent paramunity inducers, for use as drugs.
It was unexpectedly found that combining poxvirus components in the multipotent paramunity inducers according to the invention does not result in a decrease in, let alone loss of, the respective paramunizing activities of the individual poxvirus components. Instead it was seen that combining the poxvirus components derived from various poxvirus strains in the multipotent paramunity inducers according to the invention brings about not only an additive or supplementary effect, but a potentiation of the respective paramunizing action. Experiments have shown that the action of the multipotent paramunity inducers combined from poxvirus components far exceeds their respective individual actions. This phenomenon could not have been predicted, and in terms of their potency and their paramunisation-related activities it enhances these paramunity inducers as compared with conventional preparations from a single poxvirus component. Another unexpected finding is that the multipotent paramunity inducers according to the invention have virtually no immunogenic properties, but do have very strong paramunizing properties, as a result of which they can safely be administered on a repeated or continuous basis.
The invention is also based on the unexpected finding that a competitive situation exists between the epitopes of the structural proteins of the poxviruses responsible for paramunization and those responsible for immunization. The steeper the decline in activity on the part of the epitopes responsible for antigen-specific immunization, the greater is the increase in paraspecific activity. This is attested by the following two observations:
a) Attenuation over several hundred passages in cell cultures causes the immunizing properties of poxviruses to decrease, whilst the paraspecific activities not only increase but in the case of certain pox strains only appear after attenuation;
b) inactivation of the poxviruses suited to preparation of the paramunity inducers, preferably by irradiation, heat or pH action, or most preferably by chemical treatment with xcex2-propiolactone under specific conditions, causes the poxviruses to lose their immunizing properties whereas their paramunizing activities increase.
The inventive combinations of the poxvirus components are suited for use as multipotent paramunity inducers in both human and veterinary medicine.
The term xe2x80x9cpoxvirus componentxe2x80x9d, as employed in the context of this invention, covers a large number of viral structures derived from poxviruses with paramunizing properties, for example viable (i.e. capable of multiplication) or inactivated freshly isolated poxviruses, viable or inactivated recombined poxviruses derived from freshly isolated poxviruses, viable or inactivated attenuated poxviruses, viable or inactivated recombined poxviruses derived from attenuated poxviruses, the detached envelopes and cleavage products and aberrant forms of said envelopes of the poxviruses listed above, individual viral polypeptides obtained by biochemical or immunochemical methods from cultures that had been infected with the poxviruses listed above, and recombinant viral polypeptides obtained by means of prokaryotic or eukaryotic expression and at least parts of which are derived from one or more of the viral polypeptides of the poxviruses listed above.
The term xe2x80x9ccombinationxe2x80x9d, as employed in the context of this invention, covers both the mixing of two or more individual poxvirus components derived from different strains of poxvirus, and also the fact of combining a plurality of such individual poxvirus components in a polypeptide or polypeptide complex. Components derived from poxviruses, for example recombined poxviruses, individual viral polypeptides or recombinant viral polypeptides, which as a result of mutation or genetic manipulation comprise two or more polypeptide sequences derived from two or more poxvirus strains, will consequently be dealt with as though they were two or more discrete poxvirus components, irrespective of the fact that said polypeptide sequences constitute parts of a single amino acid chain. The same also goes for individual poxvirus components that are interconnected physically or chemically in any way.
The term xe2x80x9cmultipotentxe2x80x9d, as employed in the context of this invention, denotes the many and diverse potential applications of the paramunity inducers under the invention as regards prophylaxis and therapy, e.g. for the following indications:
1) infectious factor diseases and mixed infections, chronic manifestations of infectious processes, persistently relapsing infections, and bacterial and viral infections that are resistant to chemotherapy,
2) weaknesses in, or irregularities of the immune system of an organism,
3) the threat of neonatal infections,
4) adjuvant therapy in certain forms of cancer and/or prevention of metastasization,
5) regulation of homeostasis between the hormonal, circulatory, metabolic and nervous systems.
The paramunity inducers are particularly suitable for prophylactic use in the following instances:
Rapid activation of neonate immunity
Before expected stress situations (house-moving, mental stress, high-performance periods, etc.)
Before hospitalization
When there is acute danger of infection
Prevention of vaccination complications
Reducing the danger of tumours and/or metastasization
In support of bioregulation
Raising performance levels
Raising life expectancy
Regulation of homeostasis in interplay with the hormonal, circulatory, metabolic and nervous systems.
The paramunity inducers are particularly suitable for therapeutic use in the following instances:
Immune weaknesses
Virus diseases, therapy-resistant bacterial diseases, infectious factor diseases
Chronic and relapsing infections
Tumours
Convalescence
Chemotherapy and antibiotic treatment
Liver diseases of differing etiology
Chronic skin diseases
Immunopathogenic secondary diseases.
Table 1 shows the paramunizing effects of various poxvirus components and paramunity inducers in the VSV baby mouse test. The figures shown represent the efficacy indices. The procedure employed is outlined in DE-A-27 14 665.
Table 2 shows the potentiation of the paramunizing efficacy of various poxvirus strains in the VSV test as a function of the degree of attenuation involved. Inactivated virus suspensions were used.
Table 3 shows what effect attenuation has on the induction of interferon synthesis of mononuclear leucocytes of peripheral blood in various species.
Table 4 shows how the paramunizing effect of a multipotent paramunity inducer based on a recombinant poxvirus intensifies as compared with the effect of individual components.